The structure and function of the PQQ-containing quinoprotein dehydrogenases

The structure and function of the PQQ-containingquinoprotein dehydrogenases

Christopher Anthonya,*, Minakshi Ghoshb

aDivision of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton,

Southampton SO16 7PX, U.K.bLaboratory of Molecular Biophysics, University of Oxford, U.K.

Abstract

Bacterial methanol and glucose dehydrogenases containing a novel type of prosthetic group,subsequently identi®ed as pyrrolo-quinoline quinone (PQQ), were ®rst described about 30 years ago.Quinoproteins were originally de®ned as proteins containing PQQ but this de®nition has since beenbroadened to include those proteins containing other types of quinone-containing prosthetic groups, andthe X-ray structures of representatives of each type of quinoprotein have recently been published. Thisreview is mainly concerned with the structure and function of the PQQ-containing methanoldehydrogenase, whose structure has been determined at high resolution, and related proteins. Theirbasic structure consists of a `propeller' fold superbarrel made up of 8-sheet `propeller blades' which areheld together by novel tryptophan-docking motifs. In methanol dehyhdrogenase the PQQ in the activesite is coordinated to a Ca2+ ion and is maintained in position by a stacked tryptophan and a novel8-membered ring structure made up of a disulphide bridge between adjacent cysteine residues. Thisreview describes these features and discusses them in relation to previously proposed mechanisms forthis enzyme. # 1998 Elsevier Science Ltd. All rights reserved.

Keywords: Pyrrolo-quinoline quinone; Methanol dehydrogenase; Glucose dehydrogenase; Propeller fold

1. Introduction

In about 1980 the term quinoprotein was coined to include a number of bacterial

dehydrogenases which contain pyrrolo-quinoline quinone (PQQ) as their prosthetic group

(Duine et al., 1979, 1980) (Fig. 1). The name is now used more widely to include all those

Progress in Biophysics & Molecular Biology 69 (1998) 1±21

0079-6107/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved.PII: S0079-6107(97)00020-5

Pro g r e s s i n

Biophysics& MolecularBiology

PERGAMON

* Corresponding author. Tel.: 01703 594280; fax: 01703 594459; e-mail: [email protected].

enzymes whose catalytic mechanisms involve quinone-containing prosthetic groups in theiractive sites. Except for PQQ, these are derived from amino acids in the protein backbone ofthe enzyme (Fig. 1): Tryptophan tryptophylquinone (TTQ) is derived from two tryptophanresidues and occurs in bacterial amine dehydrogenases (McIntire et al., 1991; Davidson, 1993);Topa-quinone (TPQ) is a modi®ed tyrosine residue and is the prosthetic group of the copper-containing amine oxidases found in bacteria, yeasts, plants and animals (Janes et al., 1990;Klinman, 1995); and lysyl oxidase is a special type of copper-containing amine oxidase whoseprosthetic group is Lysyl tyrosylquinone (LTQ) (Wang et al., 1996).The main subjects of this review are the PQQ-containing dehydrogenases of which the

methanol dehydrogenase of methylotrophic bacteria is the best-characterized example(Anthony, 1993a; Anthony, 1996). The history of the quinoproteins began in the 1960s withthe characterisation of the novel prosthetic group of this enzyme (Anthony and Zatman, 1967),and of glucose dehydrogenase (Hauge, 1964). More than 10 years later Duine, Frank and co-workers demonstrated that it contains a quinone structure with 2 nitrogen atoms (Duine et al.,1978; Duine and Frank, 1980), and Kennard's group showed this to be pyrrolo-quinolinequinone (PQQ), by X-ray di�raction analysis (Salisbury et al., 1979). A number of otherbacterial dehydrogenases were subsequently shown to contain PQQ by the groups of Duineand Frank in Delft (Duine, 1991), and Ameyama and Adachi in Yamaguchi (Matsushita andAdachi, 1993; Matsushita et al., 1994). These quinoproteins usually catalyse the ®rst step in theoxidation of alcohols and sugars in the periplasm of bacteria, thus contributing to theformation of a protonmotive force and hence the formation of ATP (Anthony, 1993b). Theyare usually assayed with arti®cial electron acceptors such as phenazine ethosulphate (PES).The physiological electron acceptor is a soluble cytochrome c in the case of methanoldehydrogenase and some ethanol dehydrogenases; it is protein-bound haem C in thequinohaemoprotein alcohol dehydrogenases, and ubiquinone in the membrane-bound glucosedehydrogenase. For many years it was known that a divalent cation may be involved in thestructure or activity of some alcohol dehydrogenases because Ca2+ or Mg2+ are required forreconstitution of active enzyme from apoenzyme plus PQQ. By contrast, it is not possible to

Fig. 1. The prosthetic groups of quinoproteins. PQQ (pyrrolo-quinoline quinone) occurs in the dehydrogenases formethanol, higher alcohols, aldose sugars and polyvinyl alcohol, and for hydroxylation of lupanine. TTQ(tryptophan tryptophylquinone) is in bacterial amine dehydrogenases. TPQ (6-hydroxyphenylalanine quinone or

topa quinone) is in the copper-containing amine oxidases in bacteria, plants and animals. LTQ (lysinetyrosylquinone) is the prosthetic group of lysyl oxidase, a speci®c copper-containing amine oxidase occurring inanimals.

C. Anthony, M. Ghosh / Progress in Biophysics & Molecular Biology 69 (1998) 1±212

dissociate PQQ from methanol dehydrogenase without irreversible denaturation and it is onlyrelatively recently that calcium has been shown to be present and implicated in the activity ofthis enzyme (Adachi et al., 1990; Richardson and Anthony, 1992).

2. Methanol dehydrogenase

The oxidation of methanol to formaldehyde is catalysed by this enzyme in the periplasm ofmethylotrophic bacteria and is the only PQQ-containing enzyme for which a structure isavailable (Xia et al., 1992; White et al., 1993; Anthony et al., 1994; Ghosh et al., 1995;Xia et al., 1996), the highest resolution structure (1.94AÊ ) being that of the enzyme fromMethylobacterium extorquens (Ghosh et al., 1995), which is the one referred to throughout thisreview. It has an a2b2 tetrameric structure; the a-subunit containing the PQQ is 66 kDa, andthe b-subunit is very small (8.5 kDa). The subunits cannot be reversibly dissociated and nofunction has been ascribed to the small subunit. In the tetramer the two subunits ab arearranged with their pseudo 8-fold axes approximately perpendicular to each other, and thePQQ prosthetic groups are separated by about 45AÊ .

2.1. The propeller fold superbarrel structure of the a subunit of methanol dehydrogenase

The large a subunit is a superbarrel made up of eight topologically-identical four-strandedtwisted antiparallel b-sheets (W-shaped), stacked radially around a pseudo eight-fold symmetryaxis running through the centre of the subunit. This structure has been referred to as apropeller fold, each W motif representing a propeller blade and examples with four, six, sevenor eight propeller blades have been described. Haemopexin (Faber et al., 1995) and sinovialcollagenase (Li et al., 1995) have four blades; viral neuraminidase (Varghese et al., 1983) and arelated bacterial sialidase (Crennell et al., 1993) have six blades; galactose oxidase (Ito et al.,1994), methylamine dehydrogenase (Vellieux et al., 1989) and the b subunit of the G proteintransducin (Sondek et al., 1996; Lambright et al., 1996) have seven blades, while methanoldehydrogenase and nitrite reductase (Fulop et al., 1995; Baker et al., 1997) both have eightblades. It is intriguing that two of the proteins having the propeller fold structure arequinoproteins, although the related structures have no related catalytic function. Thus, inmethylamine dehydrogenase (also from methylotrophic bacteria) the TTQ prosthetic group isin the smaller subunit, the centre of the larger superbarrel being ®lled with side chains (Vellieuxet al., 1989); but, by contrast, in methanol dehydrogenase the active site containing PQQ lieswithin the large superbarrel structure.The 32 b-strands that make up the superbarrel structure in MDH are shown schematically in

Fig. 2, labelled according to the `W' motif in which they occur (1±8) and the position theytake within the motif (A±D). The sequence of the strands in the structure is the same as inthe amino acid sequence (this is also seen in other superbarrel structures), with the soleexception that the ®nal strand of the eighth motif (D8) is derived from the N-terminus, not theC-terminus. The short A strands are closest to the pseudo 8-fold axis, and the D strands areon the surface of the subunit. Fig. 2 shows that the normal twist of the b-sheet enables spaceto be e�ciently packed in the subunit. This architecture allows the large polypeptide chain to

C. Anthony, M. Ghosh / Progress in Biophysics & Molecular Biology 69 (1998) 1±21 3

be folded in a very compact form without any other typical structural domains. There is no`hole' along the pseudosymmetry axis, which is ®lled with amino acid side chains from theeight A-strands of the b-sheets which are however more hydrophilic than those on the B- andC-strands. Strand A runs more or less antiparallel to strand B, but makes only three b-type

Fig. 2. A drawing of an ab unit of MDH looking down the pseudo 8-fold axis, simpli®ed to show only the b-strands of the `W' motifs of the a-chain, and the long a-helix of the b-chain, but excluding other limited b-structuresand short a-helices (Ghosh et al., 1995). The PQQ prosthetic group is in skeletal form and the calcium ion is shownas a small sphere. The outer strand of each `W' motif is the D strand, the inner strand being the A strand. The `W'motifs are arranged in this view in an anti-clockwise manner. The exceptional motif W8 is made up of strands A±C

near the C-terminus, plus its D strand from near the N-terminus.


hydrogen bond interactions with it, all of which are close to the A/B turn. It is likely that theconformations of the A-strands are controlled by the close packing of side-chains in the verycentre of the molecule, where all eight A-strands come close together; at the point of contact®ve of the eight residues are glycine, thus facilitating close-packing. The remaining B, C and Dstrands in each motif form regular twisted b-sheets of sequences of eight to nine residues. In allmotifs the A±B and C±D corners are short, but the B±C corners are more variable and inthree cases (motifs 4, 6 and 8) they form extensions containing 24±30 residues.Cytochrome cd1 (nitrite reductase) is the only other protein, whose structure has been

determined, that has an eight bladed b-propeller structure. In this case the centre of the barrelcontains haem d instead of PQQ. Remarkably, the b-propeller domain of cytochrome cd1proves to be closely superimposable on that of methanol dehydrogenase although the twopolypeptides have no sequence identity and they use two di�erent folding patterns to bind the®rst and eighth blades together (Baker et al., 1997). In methanol dehydrogenase this closure ofthe ring is achieved by forming the eighth blade from three b-strands from the C-terminus plusone strand provided by the N terminus. By contrast, in cytochrome cd1 the eighth blade isformed from three b-strands from the N-terminus plus one strand from the C-terminus. Bothof these folding patterns have been observed previously in other propeller proteins with fewerthan eight blades. At present no sequences can be identi®ed which can be used to predictpropeller structures or to determine which folding pattern for closure will be present. Murzin(1992) has published a valuable discussion of the principles involved in propeller assembly ofb-sheets.

2.2. The tryptophan-docking interactions in the a-subunit of methanol dehydrogenase

A series of tryptophan-docking interactions between the b sheet propeller blades makeplanar, stabilising girdles around the periphery of the subunits (Ghosh et al., 1995; Xia et al.,1996) (Fig. 3). This method of stabilising protein structures has not been described previouslyand is therefore described here in some detail. The interactions occur by way of 11-residueconsensus sequences in the C/D region of all `W' motifs except number 8 (Fig. 4). The relevantcharacteristics of the tryptophan residues are their planar conjugated rings, and their ability toact as a hydrogen bond donor through the indole ring NH group (Fig. 5). The tryptophan atposition 11 is stacked between the alanine at position 1 of the same motif (Wn) and the peptidebond between residue 6 and the invariant glycine at position 7 of the next motif (Wn + 1). Thesame tryptophan is also H-bonded between its indole NH and the main chain carbonyl ofresidue 4 in the next motif (Wn + 1). The third type of interaction involving the conservedtryptophans is a b sheet hydrogen bond between its carbonyl oxygen and the backbonenitrogen of position 1 (usually alanine) of the same motif.Exceptions to these interactions with tryptophan occur in W1 and W7. In W1 the glycine at

position 7 is replaced in the stacking interaction by proline at position 6; and in W7 thealanine at position 1 (Ala498) does not form the expected interaction with Trp508. There aretwo exceptions to tryptophan in position 11. Tryptophan is replaced by Ser347 in W5 whichmakes a hydrogen bond with the peptide carbonyl of residue 4 in motif W6. In the otherexception, Phe292 replaces tryptophan in W4 and makes only stacking interactions, with theusual glycine peptide bond of W5 on one side, and a second glycine peptide bond in position 1


of W4. Motif W8 is exceptional in having no consensus sequence (except for Trp44) and sothere is no glycine to interact with the tryptophan in the previous motif (Trp508); in its placethis tryptophan forms a hydrophobic interaction with the side chain of Leu40 and in theprevious motif. The carbonyl of this leucine (position 7) replaces the usual carbonyl at position4 in forming a hydrogen bond with the tryptophan indole NH. The 11-residue motif isextended in many cases by two further residues on the C and D strands which are joined bymain chain hydrogen bonds (Xia et al., 1996). The PQQ-containing dehydrogenases for glucose(GDH) and alcohol (ADH) show the greatest similarity to the MDH in the sequences whichform the W motifs. This has facilitated modelling of the structure of these proteins which hassuggested that they have an almost identical tryptophan docking motif at the C/D corners asindicated for MDH (Fig. 4).No similar tryptophan docking motifs are present in the 7-bladed superbarrel proteins

galactose oxidase and methylamine dehydrogenase, or in the 8-bladed nitrite reductase(cytochrome cd1) (Fulop et al., 1995; Baker et al., 1997) in which the centre of the superbarrelprovides a pocket for the d1 haem. This is particularly remarkable for the nitrite reductase

Fig. 3. The girdle of tryptophan residues involved in docking the b-sheets together (Ghosh et al., 1995). Thetryptophan residues involved in docking are shown in space®ll mode and the rest of the chain as backbone.ThePQQ prosthetic group is in skeletal form and the calcium ion is shown as a small sphere.


because the structure of the superbarrel domain of this enzyme can be superimposed on that ofmethanol dehydrogenase.The subunits in methanol dehydrogenase contact one another in the region containing the

D-strands of the seventh and eighth W motifs, associated over a large planar interfacecontaining many hydrophobic and hydrophilic side chain interactions. These are augmented bythe ten C-terminal residues of the a-chains which form extensions which associate with thesymmetry-related subunit, again through hydrophobic and hydrophilic interactions.

2.3. The structure of the b-subunit of methanol dehydrogenase

The b subunit (Fig. 2) is most unusual as it forms a very extended structure having nohydrophobic core. The N-terminal 30 residues, which include one intrachain disulphide bridge(Cys6±Cys12) and a proline-rich segment (residues 14±20), is folded in a series of open turns.The C-terminal 54 residues, rich in charged residues, form a single straight a-helix of 30residues in seven turns followed by a C-terminal segment folded back on the helix. Overall, theb-chain forms a planar `J' shaped unit, with the long a-helix as its stem, which hooks over theglobular a-subunit. Although some hydrophobic interactions occur between the a and b subunits,

Fig. 4. The consensus sequences in the tryptophan docking motif (Ghosh et al., 1995). This occurs at the C/Dcorners at the end of the C strands and the beginning of the D strands of each W motif; there are no loops betweenthese strands. The C/D corners are best characterised as 4-residue (b) turns or 5-residue turns (comprising residues3±6 or 3±7 respectively). Consensus sequences are also included for the quinohaemoprotein alcohol dehydrogenase

(ADH) and glucose dehydrogenase, which is a membrane quinoprotein (GDH).


ion-pair interactions are predominant, as expected from the fact that 40% of the b-chainresidues are charged. The b-chain makes contact with the edges of the W1±W4 motifs of the a-chain with ion pair interactions involving Glu148, Glu193, Arg197, Lys236, Glu267 andGlu301 on the a-chain, with Lys16, Glu48, Arg50, Arg54, and Lys59 of the b-chain. In theabsence of any other obvious function for this unusual subunit, it has been suggested that itacts to stabilise the folded form of the large a-chain. The absence of b-subunits in the otherPQQ-containing quinoproteins, however, perhaps indicates that it has a more speci®c(unknown) function.

2.4. The novel disulphide ring structure in the active site of methanol dehydrogenase

Within the a subunit there is a remarkable novel structure derived by formation of adisulphide bridge between adjacent cysteine residues (Cys103±Cys104); the result is a strained8-membered ring (Fig. 6). Although such a disulphide bridge has been proposed to be presentin the active site of the acetylcholine receptor (Kao and Karlin, 1986) and has been seen in thestructure of an inactive, oxidised, form of mercuric ion reductase (Schiering et al., 1991), this isthe ®rst time that this structure has been seen in an active enzyme. It has been predicted that ifsuch a structure should unexpectedly exist in a protein then a strained ring would be producedin which a normal planar trans peptide bond would not be possible, and that a cis peptide

Fig. 5. Part of a typical tryptophan-docking motif. This shows the interactions of Trp 145 (in W2) with Ala135(also in W2), and the interaction with the plane of the peptide bond between Thr191 and Gly192 (in W3). For

clarity the remaining residues of the two motifs are omitted, and the side chains are omitted from all residues exceptfor Trp145 and Ala135.


bond would be more likely (Ramachandran and Sasisekharan, 1968; Chandrasekaran andBalasubramanian, 1969). In our 1.94AÊ structure of the enzyme it is clearly seen that thepeptide bond is, as predicted, non-planar, but it is in the trans con®guration. The o angle is1458, giving a distortion from planarity of 358. All of the other bond lengths and bond anglesin the ring are standard values, including the distance between the sulphur atoms (2.06 AÊ ); thedistortion in the polypeptide chain within the disulphide ring structure is thus di�erent fromthat observed in the isolated Cys±Cys dipeptide model compounds (Capasso et al., 1977; Mez,1974; Sukumaran et al., 1991).

2.5. The active site of methanol dehydrogenase

The active sites within each a subunit occur on the pseudo 8-fold symmetry axis, at the endof the superbarrel structure containing the loops between the B and C strands of motifs W6and W8 which fold over the end of the superbarrel to enclose the active site chamber. Access isby way of a shallow funnel made up of hydrophobic surface residues (Anthony et al., 1994)leading to a narrow entrance to the chamber containing the non-covalently bound PQQ, abound Ca2+ ion, the novel disulphide ring structure and a potential active site base (Asp-303).There is no obvious interaction between the two active sites in the two a subunits, and the bsubunits make no direct contribution to the structure of the active sites.The PQQ is held in place by way of two non-polar axial interactions, the PQQ ring being

sandwiched between the indole ring of Trp243 and the disulphide ring structure (Fig. 6).The indole ring is within 158 of coplanarity with the PQQ ring and in contact with it and, on

Fig. 6. The novel disulphide ring in the active site of methanol dehydrogenase (Ghosh et al., 1995). The ring is

formed by disulphide bond formation between adjacent cysteine residues. The PQQ is `sandwiched' between thisring and the tryptophan that forms the ¯oor of the active site chamber. The calcium ion is coordinated between theC-9 carboxylate, the N-6 of the PQQ ring and the carbonyl oxygen at C-5. The oxygen of the C-4 carbonyl appears

to be out of the plane of the ring.


the opposite side the two sulphur atoms of the disulphide bridge, are within 3.75AÊ of the planeof PQQ. In addition to these axial interactions, many amino acid residues are involved inequatorial interactions with the substituent groups of the PQQ ring system. These areexclusively hydrogen-bond and ion-pair interactions involving residues mostly on the A strandsof the `W' motifs (Fig. 7). Although the number of polar groups involved might indicate at®rst sight that the environment of the PQQ is polar, earlier ENDOR experiments hadpreviously indicated a relatively hydrophobic environment for PQQ and a closer look at theinteractions con®rms this. An oxygen of the 9-carboxyl forms a salt bridge with Arg109 andboth groups are shielded from bulk solvent by the disulphide. The carboxyl group of Glu155and a 2-carboxyl oxygen of PQQ are also shielded from solvent and it is probable that at leastone is protonated, their interaction thus being stabilised through hydrogen bond formation.Of particular interest are the interactions of the C4 and C5 oxygen atoms; whereas the C5

oxygen is in the plane of the PQQ ring system, the C4 oxygen appears to be out of the planeby about 408 (Ghosh et al., 1995). This is inconsistent with PQQ being in either the oxidisedquinone form or the reduced quinol form; this is consistent with the observation that, whenisolated, the PQQ is always in the semiquinone state (Duine and Frank, 1980; Frank et al.,

Fig. 7. The equatorial interactions of PQQ and the coordination of Ca2+ in the active site of methanoldehydrogenae (Ghosh et al., 1995). This ®gure also shows Asp303, which is likely to act as a base, and Arg331which may also be involved in the mechanism. Figure 6 shows the axial interactions that are also involved in

holding PQQ in place in the active site.


1988; Dijkstra et al., 1989; Richardson and Anthony, 1992; Avezoux et al., 1995). The C4 andC5 oxygens are hydrogen bonded by the NH1 and NH2 atoms respectively of Arg331, and inaddition the C4 oxygen makes a longer hydrogen bond interaction with the amide NH2 ofAsn394 whose amide CO is hydrogen bonded to its own main chain NH group. It is notknown if the bonding of the C4 and C5 oxygen atoms is maintained in the fully oxidisedquinone and fully reduced quinol forms of the prosthetic group, in which the C4 and C5oxygen atoms are likely to be in the plane of the ring. As well as its hydrogen bonding to thePQQ, Arg331 also makes hydrogen bonds between its NH2 and the carboxylate and main-chain carbonyl of Asp303. The two side chains lie side by side, permitting free access to thecarboxyl group of Asp303, which is the most likely candidate for the base required by somecatalytic mechanisms previously proposed for MDH (Anthony, 1996, 1997).A Ca2+ ion is clearly seen in the active site, con®rming predictions that it is likely to be

intimately related to the prosthetic group (Richardson and Anthony, 1992). The co-ordinationsphere of the calcium ion in the active site contains PQQ and protein atoms (Fig. 7), includingboth oxygens of the carboxylate of Glu177 and the amide oxygen of Asn261. The PQQ atomsinclude the C5 quinone oxygen, one oxygen of the C7 carboxylate and, surprisingly, the N6ring atom which is only 2.45AÊ from the metal ion. The ®ve oxygen ligands have distancesfrom the Ca2+ of 2.4±2.8AÊ .The position of the substrate in the active site has not been unequivocally determined; the

one or two solvent molecules which might occupy the same space as the substrate alcoholgroup are in slightly di�erent locations in the two available structures (Ghosh et al., 1995; Xiaet al., 1996). Immediately adjacent to the more polar region containing the active site basethere is a hydrophobic cavity, which could accommodate a small alkyl group, bounded by twotryptophans, a leucine and the disulphide ring. This raises the problem that this enzyme has abroad substrate speci®city, and can oxidise primary alcohols including relatively largesubstrates such as pentanol and cinnamyl alcohol; it is not immediately obvious how thesesubstrates could readily gain access to the active site and understanding of this awaits solutionof a structure containing one of these larger substrates.

2.6. The mechanism of methanol dehydrogenase

The enzyme catalyses a Ping±Pong reaction, consistent with reduction of PQQ by substrateand release of product, followed by two sequential single-electron transfers to the cytochromecL, during which the PQQH2 is oxidised back to the quinone by way of the free radicalsemiquinone (Duine and Frank, 1980; Frank et al., 1988; Dijkstra et al., 1989). The rate-limiting step is the conversion of the oxidised complex, containing the substrate, into thereduced enzyme plus product, and is the only step requiring the activator ammonia.The C-5 carbonyl of isolated PQQ is very reactive towards nucleophilic reagents, such as

methanol, leading to the obvious conclusion that a covalent PQQ-substrate complex may beimportant in the reaction mechanism. Support for this has come from the reaction of MDHwith cyclopropanol which gives a C-5 propanal adduct, indicating that the mechanism consistsof proton abstraction by a base, giving a ring-opened carbanion, which then attacks theelectrophilic C-5 of PQQ (Frank et al., 1989a,b). It was suggested that during oxidation ofmethanol a similar proton abstraction must occur, followed by formation of a carbon/oxygen


bond to give a hemiketal intermediate. It is probable that Asp-303 (Fig. 7) provides thecatalytic base which initiates the reaction by abstraction of a proton from the alcohol substrate(Fig. 8). In the mechanism shown in Fig. 7 the oxyanion produced by proton abstractionattacks the electrophilic C-5, leading to formation of the proposed hemiketal intermediate, thesubsequent reduction of the PQQ with release of product aldehyde being facilitated by priorionization of the hemiketal complex which might involve the pyrrole N atom. An alternativemechanism, is a simple acid/base-catalysed hydride transfer in which Asp-303 again providesthe base and Ca2+ acts again as a Lewis acid (Fig. 9). The large deuterium isotope e�ect(about 6) observed during the reductive phase of the reaction is consistent with eithermechanism; in both cases the step a�ected will be the breaking of the C±H bond, and it is thisstep that is a�ected by the activator ammonia, although the mechanism of this activation is notunderstood (Frank et al., 1988; Goodwin and Anthony, 1996). Recent studies using PQQanalogues bonded to Ca2+ in organic solvents have provided supporting evidence formechanisms involving hemiketal formation (Fig. 8) (Itoh et al., 1997).In the mechanisms described here the Ca2+ ion is given a role in addition to a structural

role in maintaining PQQ in an active con®guration; it is proposed that the Ca2+ acts as aLewis acid by way of its coordination to the C-5 carbonyl oxygen of PQQ thus stabilisingthe electrophilic C-5 for attack by an oxyanion or hydride (Anthony et al., 1994; Anthony,

Fig. 8. Mechanism for methanol dehydrogenase involving formation of a hemiketal intermediate (Anthony, 1996,

1997). It is suggested that the base (Asp303) abstracts a proton from methanol and that the Ca2+ ion facilitatesattack by the resulting oxyanion on the electrophilic C-5, to give the hemiketal from which the methyl proton isabstracted; this is facilitated by ionizationof the C-4 carbonyl oxygen which is made possible by the pyrrole nitrogenatom. The oxidative part of the cycle involves electron transfer to cytochrome cL or an arti®cal dye electron

acceptor.


1996, 1997). It is also possible that the Ca2+ ion coordinates to the substrate oxygenatom. The role of Ca2+ in the mechanism has been given support by a study of a Sr2+-containing methanol dehydrogenase produced by growing bacteria in a high concentration ofSr2+ (Harris and Davidson, 1994), and by investigations using an active enzyme containingBa2+ instead of Ca2+ (Goodwin and Anthony, 1996). This is the ®rst example of an enzymein which barium plays an active catalytic role; the modi®ed enzyme has a relatively lowa�nity for methanol (Km, 3.4 mM instead of 10 mM), and for its activator ammonia, but itsactivation energy is half (and its Vmax twice) that of the normal Ca2+ enzyme. We havesuggested that this may be due to a change in conformation at the active site, leading to adecrease in free energy of binding and hence to a decrease in activation energy (Goodwin andAnthony, 1996). It should be noted that, as an alternative to Ca2+ acting as activator of theC5 atom, it has been suggested that Arg-331, which hydrogen bonds to the C5 oxygen, mightbe involved as an electrophile leading to build up of positive charge density on this oxygen(Xia et al., 1996).

2.7. The reaction of methanol dehydrogenase with its electron acceptor, cytochrome cL

Cytochrome cL is a speci®c large acidic c-type cytochromes (Nunn and Anthony, 1988;Anthony, 1992; Chen et al., 1994). There is considerable evidence that the dehydrogenase andcytochrome cL `dock' together, initially by electrostatic interactions between a small number oflysyl residues on the dehydrogenase and carboxylates on the cytochrome (Dijkstra et al., 1989;Anthony, 1992; Chan and Anthony, 1991; Cox et al., 1992). A study of this initial interactioncon®rmed the role of electrostatic inter actions but surprisingly showed that it is not inhibitedby 50 mM-EDTA which is su�cient to inhibit the overall electron transfer process between theproteins (Dales and Anthony, 1995). It is possible, therefore, that EDTA inhibits by binding tonearby lysyl residues, thus preventing movement of the `docked' cytochrome to its optimalposition for electron transfer, which probably involves interaction with the hydrophobic funnelin the surface of the dehydrogenase (Harris and Davidson, 1993; Anthony et al., 1994; Ghoshet al., 1995; Dales and Anthony, 1995; Harris et al., 1994).

Fig. 9. An alternative hydride transfer mechanism. The initial proton abstraction is the same but the electrophilic C-5 is involved directly in removal of the methyl hydrogen as hydride. This mechanism was adapted from thosepreviously published (Anthony et al., 1994a) in order to emphase the probable double involvement of the active site

base (Asp303). The oxidative phase is the same as in Figure 8.


Electron transfer from the quinol form of PQQ to the cytochrome electron acceptor occursin two single electron transfer stepsÐthe semiquinone form of PQQ being produced after the®rst of these transfers (Fig. 8). The protons are released from the reduced PQQ into theperiplasmic space thus contributing to the protonmotive force (Anthony, 1988, 1993b). Anobvious candidate as an intermediary in this process is the novel disulphide bridge betweenadjacent cysteines in the active site. This possibility appeared to be supported by thedemonstration that this novel structure is very readily reduced with dithiothreitol, yieldingenzyme that is inactive with cytochrome. However, reaction of the inactive reduced enzymewith iodoacetate produces carboxymethylated cysteine residues that would not be availableto take part in oxidation/reduction reactions, and yet this carboxymethylation leads to re-formation of active enzyme (Avezoux et al., 1995). This type of disulphide ring structure hasnot been observed previously in an active enzyme and its rarity would suggest some specialbiological function. It is not present in the quinoprotein glucose dehydrogenase in whichelectrons are transferred to membrane ubiquinone from the quinol PQQH2, and in which thesemiquinone free radical is unlikely to be involved as a stable intermediate. It has beensuggested, therefore, that this novel structure might function in the stabilization or protectionfrom solvent at the entrance to the active site of the free radical PQQ semiquinone in methanoldehydrogenase (Avezoux et al., 1995).

3. The PQQ-containing dehydrogenase for alcohols

There are three types of PQQ-containing alcohol dehydrogenase that are distinct frommethanol dehydrogenase (Matsushita and Adachi, 1993) but they all contain a calciumion, and the reductive parts of their mechanisms are likely to be similar. The ®rst type, suchas that in Pseudomonas aeruginosa is almost identical except for its substrate speci®city(Schrover et al., 1993). The other two types are quinohaemoproteins, having an in-builtelectron acceptor in the form of haem C which occurs on a C-terminal extension of theprimary sequence. This is illustrated in Fig. 10, which also shows the membrane glucosedehydrogenase with its N-terminal additional sequence likely to be involved in binding theenzyme in the membrane.The quinohaemoprotein alcohol dehydrogenase from acetic acid bacteria is membrane-bound

and contains three types of subunit but no subunit equivalent to the small n subunit ofmethanol dehydrogenase (Matsushita and Adachi, 1993; Matsushita et al., 1994). The primarysequence of the catalytic subunit shows an N-terminal region (600 residues) with an additionalC-terminal extension containing a haem-binding site (Inoue et al., 1989, 1990). In the N-terminal region there was 31% identity to the sequence of methanol dehydrogenase and it waspossible to model its structure using the coordinates of methanol dehydrogenase (Fig. 11)(Cozier et al., 1995). Although there are considerable di�erences in the external loops,particularly those involved in the formation of the shallow funnel leading to the active site, theactive site region was highly conserved, including the tryptophan and the disulphide ring onopposite sides of the plane of the PQQ, and also most of equatorial coordinations to thePQQ (Fig. 7). Especially important with respect to the mechanism was conservation of theactive site base (Asp-303 in methanol dehydrogenase) and all the coordinations to the calcium


ion. This suggests that the mechanism of this alcohol dehydrogenase is essentially similar tothat of the methanol dehydrogenase. Comparison of the protein sequence of the solublequinohaemoprotein ethanol dehydrogenase from Comomonas testosteroni leads to a similarconclusion for that enzyme (Stoorvogel et al., 1996).

4. The PQQ-containing membrane-bound glucose dehydrogenase

The membrane-bound glucose dehydrogenase catalyses the oxidation of the pyranose formof D-glucose (at the C1 position) and other monosaccharides to the lactone. The reactionoccurs in the periplasm and the electron acceptor is ubiquinone in the membrane (Matsushitaet al., 1986, 1989). It is an intrinsic monomeric membrane protein (about 87 kDa) in which adivalent cation is necessary for reconstitution of active enzyme from apoenzyme plus PQQ.The N-terminal region (residues 1±154) forms a membrane anchor with 5 trans-membranesegments, and this region is likely to contain the ubiquinone-binding site. The remainingperiplasmic region (residues 155±796) shows 26% identity of sequence to that of the a-subunitof methanol dehydrogenase and it has been possible to model its structure using thecoordinates of methanol dehydrogenase (Fig. 11) (Cozier and Anthony, 1995). In the modelstructure, the novel disulphide ring is replaced by a histidine residue which maintains theposition of PQQ in the active site, consistent with the previous demonstration that a histidineresidue is essential for binding PQQ (Imanaga, 1989) (Fig. 12). There are fewer equatorialinteractions between the protein and PQQ (Fig. 13), perhaps explaining why it is possible toe�ect the reversible dissociation of PQQ from the glucose dehydrogenase but not from

Fig. 10. Sequence alignment of quinoprotein dehydrogenases. Each `W' is a 4-stranded b-sheet (or propeller blade).These are the regions showing greatest similarity of sequence between the quinoproteins. There are many loops

between, and within, the b-sheets which show least similarity. for example, there is a long region with littleconservation of sequence (including a large loop) between the end of the D-strand in W5 and the end of the D-strand of W6. The highly conserved region between strand-A in W7 and the end of strand-B in W8 was originally

proposed to be a PQQ-binding domain; this is not the case and the reason for the high level of conservation is notunderstood.


Fig. 11. Schematic representation of the backbone of glucose dehydrogenase (GDH), methanol dehydrogenase(MDH) and alcohol dehydrogenase (ADH) showing their major secondary structure. The MDH was determined by

X-ray di�raction (Ghosh et al., 1995).The model GDH structure is of the C-terminal section of the membrane-bound GDH (residues 155±796) (Cozier and Anthony, 1995). The model ADH structure is of the N-terminal regionof the quinohaemoprotein subunit I of the membrane complex isolated from acetic acid bacteria (residues 1±590)(Cozier et al., 1995). The prosthetic group is shown as a ball and stick structure, and the Ca2+ as a van der Waal's

sphere. The major loops are in black. The position marked (h) shows the position where residues 497±579 (GDH)would join the main superbarrel structure. These residues are not present in MDH or ADH and the sequence onGDH is too long to model.


methanol dehydrogenase (Ameyama et al., 1985; Matsushita and Adachi, 1993). By analogywith the methanol dehydrogenase structure Asp466 is likely to be involved in base catalysis.One clear di�erence is that there is more `space' in the glucose dehydrogenase active site,perhaps to accommodate the larger substrate, and the Arg331 in MDH which may play a rolein catalysis is replaced by Lys493 in glucose dehydrogenase (Cozier and Anthony, 1995). Theligation of the (presumed) calcium is similar, suggesting that it plays a similar role in the twoenzymesÐthat of a Lewis acid through coordination to the C-5 carbonyl oxygen, thusstabilising the electrophilic C-5 of PQQ. The proposed active site base is conserved, suggestingthat the reaction is initiated by abstraction of a proton from the anomeric hydroxyl of thepyranose ring. This would be followed by attack by the resulting oxyanion to form a hemiketalintermediate; or attack by a hydride from the glucose oxyanion, leading directly to formationof the lactone and the quinol form of PQQ. When active holoenzyme is reconstituted frominactive apoenzyme plus PQQ a divalent metal is required; for this purpose Mg2+ is usuallybetter than Ca2+ (Ameyama et al., 1985; Buurman et al., 1994). If this reconstitution leads toincorporation of Mg2+ instead of Ca2+ at the active site then this might imply some di�erencein mechanism between the glucose dehydrogenase and methanol dehydrogenase.The oxidative half reaction of glucose dehydrogenase is completely di�erent from that in

methanol dehydrogenase in which there must be two single electron transfers to two separatecytochrome molecules. By contrast, in glucose dehydrogenase two hydrogen atoms must betransferred to the acceptor ubiquinone. Although this may also involve transfer of the electronsone at a time it is not necessary for a stable semiquinone to be formed, and indeed nosemiquinone has ever been observed in glucose dehydrogenase. The active site funnel is nothydrophobic and there is no suggestion from the model structure or from the primary sequencethat there is a hydrophobic region of the protein that could interact with the membrane exceptthe N-terminal transmembrane segments.

Fig. 12. Comparison of the stacking interactions of the PQQ in MDH and the model GDH. In MDH the PQQ isstacked between the coplanar Trp-243 and the disulphide ring system of Cys103 and Cys104 (Ghosh et al., 1995). In

GDH the coplanar tryptophan is retained (Trp-404) but the disulphide is not conserved. Instead, His-262 mayperform a similar role in helping to bind the PQQ into the active site region.


5. Future prospects

Now that the structure of the PQQ-containing quinoprotein methanol dehydrogenase has beendetermined, the most important remaining questions concern details of the mechanism of itsaction, including the role of the activator ammonia, the `docking' system for interaction with itsspeci®c cytochrome, and determination of the route for electron transfer within the enzyme andthe transfer of electrons to the cytochrome. Similar studies will be extended to the related alcoholand glucose dehydrogenases. The approaches to address these questions involve analysis ofstructures of multiprotein complexes, and of proteins containing activator and substrate, and theuse of site directed mutagenesis together with kinetic and structural studies of the mutant proteins.

Fig. 13. The coordination of Ca2+ and the bonding of PQQ in the active site of the model glucose dehydrogenase.Of the equatorial interactions with PQQ the signi®cant di�erences between the two enzymes are that residues Ser-174, Arg-331 and Trp-476 of MDH are replaced by Val-352, Lys-493 and Leu-712 in GDH; this results in fewer H-

bonds to the PQQ in GDH. Ca2+ is included in the model GDH, although this may be replaced by Mg2+ in theGDH from some bacteria. By analogy with the mechanism proposed for MDH (Anthony, 1993a, 1996), Asp-466may act as a base, initiating the reaction by abstraction of a proton from glucose; in this mechanism the Ca2+ actsas a Lewis acid, co-ordinating with the C-5 carbonyl oxygen, stabilising the electrophilic C-5 carbon of PQQ.


Acknowledgements

We should like to thank The EPSRC, BBSRC, The Wellcome Trust and the Royal Societyfor support of our work on quinoproteins, and the Department of Biochemistry, SriVenkateswara University (Tirupati) and The Indian Institute of Science (Bangalore) for theirhospitality during production of this review.

References

Adachi, O., Matsushita, K., Shinagawa, E., Ameyama, M., 1990. Calcium in quinoprotein methanol dehydrogenase can be replaced by

strontium. Agric. Biol. Chem. 54, 2833±2837.

Ameyama, M., Nonobe, M., Hayashi, M., Shinagawa, E., Matsushita, K., Adachi, O., 1985. Mode of binding of pyrroloquinoline

quinone to apo-glucose dehydrogenase. Agric. Biol. Chem. 49, 1227±1231.

Anthony, C. (1988) Quinoproteins and energy transduction. In Bacterial Energy Transduction, ed. C. Anthony, pp. 293±316. Academic

Press, London.

Anthony, C., 1992. The c-type cytochromes of methylotrophic bacteria. Biochim. Biophys. Acta 1099, 1±15.

Anthony, C. (1993a) Methanol dehydrogenase in Gram-negative bacteria. In Principles and applications of quinoproteins, ed. V. L.

Davidson, pp. 17±45. Marcel Dekker, New York.

Anthony, C. (1993b) The role of quinoproteins in bacterial energy transduction. In Principles and applications of quinoproteins, ed. V.

L. Davidson, pp. 223±244. Marcel Dekker, New York.

Anthony, C., 1996. Quinoprotein-catalysed reactions. Biochem. J. 320, 697±711.

Anthony, C. (1997) Quinoprotein-catalysed reactions. In Comprehensive biological catalysis; a mechanistic reference. Academic Press,

London.

Anthony, C., Ghosh, M., Blake, C. C. F., 1994. The structure and function of methanol dehydrogenase and related PQQ-containing

quinoproteins. Biochem. J. 304, 665±674.

Anthony, C., Zatman, L. J., 1967. The microbial oxidation of methanol: the prosthetic group of alcohol dehydrogenase of

Pseudomonas sp. M27; a new oxidoreductase prosthetic group. Biochem. J. 104, 960±969.

Avezoux, A., Goodwin, M. G., Anthony, C., 1995. The role of the novel disulphide ring in the active site of the quinoprotein methanol

dehydrogenase from Methylobacterium extorquens. Biochem. J. 307, 735±741.

Baker, S. C., Saunders, N. F. W., Willis, A. C., Ferguson, S. J., Hajdu, J., Fulop, V., 1997. Cytochrome cd1 structure: unusual haem

environments in a nitrite reductase and analysis of factors contributing to b-propeller folds. J. Mol. Biol. 269 (Abstract), 440±455.

Buurman, E. T., Tenvoorde, G. J., Demattos, M. J. T., 1994. The physiological function of periplasmic glucose oxidation in phosphate-

limited chemostat cultures of Klebsiella pneumoniae NCTC 418. Microbiology, U.K. 140, 2451±2458.

Capasso, S., Mattia, C., Mazzarella, L., 1977. Structure of a cis-peptide unit: molecular conformation of the cyclic disulphide L-cystei-

nyl-L-cysteine. Acta Cryst. B33, 2080±2083.

Chan, H. T. C., Anthony, C., 1991. The interaction of methanol dehydrogenase and cytochrome cL in the acidophilic methylotroph

Acetobacter methanolicus. Biochem. J. 280, 139±146.

Chandrasekaran, R., Balasubramanian, R., 1969. Stereochemical studies of cyclic peptides. VI. Energy calculations of the cyclic dipep-

tide cysteinyl-cysteine. Biochim. Biophys. Acta 188, 1±9.

Chen, L. Y., Durley, R. C. E., Mathews, F. S., Davidson, V. L., 1994. Structure of an electron transfer complexÐmethylamine dehy-

drogenase, amicyanin, and cytochrome-c(551I). Science 264, 86±90.

Cox, J. M., Day, D. J., Anthony, C., 1992. The interaction of methanol dehydrogenase and its electron acceptor, cytochrome cL, in the

facultative methylotroph Methylobacterium extorquens AM1 and in the obligate methylotroph Methylophilus methylotrophus.

Biochim. Biophys. Acta 1119, 97±106.

Cozier, G. E., Giles, I. G., Anthony, C., 1995. The structure of the quinoprotein alcohol dehydrogenase of Acetobacter aceti modelled

on that of methanol dehydrogenase from Methylobacterium extorquens. Biochem. J. 307, 375±379.

Cozier, G. E., Anthony, C., 1995. Structure of the quinoprotein glucose dehydrogenase of Escherichia coli modelled on that of metha-

nol dehydrogenase from Methylobacterium extorquens. Biochem. J. 312, 679±685.

Crennell, S. J., Garman, E. F., Laver, W. G., Vimr, E. R., Taylor, G. L., 1993. Crystal structure of a bacterial sialidase (from

Salmonella typhimurium LT2) shows the same fold as an in¯uenza virus neuraminidase. Proc. Natl. Acad. Sci. USA 90, 9852±9856.

Dales, S. L., Anthony, C., 1995. The interaction of methanol dehydrogenase and its electron acceptor. Biochem. J. 312, 261±265.

Davidson, V. L. (1993) Principles and applications of quinoproteins. Marcel Dekker, New York.


Dijkstra, M., Frank, J., Duine, J. A., 1989. Studies on electron transfer from methanol dehydrogenase to cytochrome cL, both puri®ed

from Hyphomicrobium X. Biochem. J. 257, 87±94.

Duine, J. A., Frank, J., Westerling, J., 1978. Puri®cation and properties of methanol dehydrogenase from Hyphomicrobium X.

Biochim. Biophys. Acta 524, 277±287.

Duine, J. A., Frank, J., van Zeeland, J. K., 1979. Glucose dehydrogenase from Acinetobacter calcoaceticus. FEBS Lett. 108, 443±446.

Duine, J. A., Frank, J., Verwiel, P. E. J., 1980. Structure and activity of the prosthetic group of methanol dehydrogenase. Eur. J.

Biochem. 108, 187±192.

Duine, J. A., 1991. QuinoproteinsÐenzymes containing the quinonoid cofactor pyrroloquinoline quinone, topaquinone or trypto-

phan- tryptophan quinone. Eur. J. Biochem. 200, 271±284.

Duine, J. A., Frank, J., 1980. The prosthetic group of methanol dehydrogenase: puri®cation and some of its properties. Biochem. J.

187, 221±226.

Faber, H. R., Groom, C. R., Baker, H. M., Morgan, W. T., Smith, A., Baker, E. N., 1995. 1.8 Angstrom crystal structure of the C-

terminal domain of rabbit serum haemopexin. Structure 3, 551±559.

Frank, J., Dijkstra, M., Duine, A. J., Balny, C., 1988. Kinetic and spectral studies on the redox forms of methanol dehydrogenase from

Hyphomicrobium X. Eur. J. Biochem. 174, 331±338.

Frank, J., Dijkstra, M., Balny, C., Verwiel, P. E. J. and Duine, J. A. (1989a) Methanol dehydrogenase: mechanism of action. In PQQ

and Quinoproteins, eds. J. A. Jongejan and J. A. Duine, pp. 13±22. Kluwer Academic Publishers, Dordrecht.

Frank, J., van Krimpen, S. H., Verwiel, P. E. J., Jongejan, J. A., Mulder, A. C., Duine, J. A., 1989b. On the mechanism of inhibition of

methanol dehydrogease by cyclopropane-derived inhibitors. Eur. J. Biochem. 184, 187±195.

Fulop, V., Moir, J. W. B., Ferguson, S. J., Hajdu, J., 1995. The anatomy of a bifunctional enzyme: structural basis for reduction of

oxygen to water and synthesis of nitric oxide by cytochrome cd1. Cell 81, 369±377.

Ghosh, M., Anthony, C., Harlos, K., Goodwin, M. G., Blake, C. C. F., 1995. The re®ned structure of the quinoprotein methanol de-

hydrogenase from Methylobacterium extorquens at 1.94 A. Structure 3, 177±187.

Goodwin, M. G., Anthony, C., 1996. Characterisation of a novel methanol dehydrogenase containing barium instead of calcium.

Biochem. J. 318, 673±679.

Harris, T. K., Davidson, V. L., Chen, L. Y., Mathews, F. S., Xia, Z. X., 1994. Ionic strength dependence of the reaction between

methanol dehydrogenase and cytochrome c-551i: evidence of conformationally coupled electron transfer. Biochemistry 33,

12600±12608.

Harris, T. K., Davidson, V. L., 1993. Binding and electron transfer reactions between methanol dehydrogenase and its physiologic

electron acceptor cytochrome-c-551iÐa kinetic and thermodynamic analysis. Biochemistry 32, 14145±14150.

Harris, T. K., Davidson, V. L., 1994. Replacement of enzyme-bound calcium with strontium alters the kinetic properties of methanol

dehydrogenase. Biochem. J. 300, 175±182.

Hauge, J. G., 1964. Glucose dehydrogenase of Bacterium anitratum: an enzyme with a novel prosthetic group. J. Biol. Chem. 239,

3630±3639.

Imanaga, Y. (1989) Investigations on the active site of glucose dehydrogenase from Pseudomonas ¯uorescens. In PQQ and

Quinoproteins, eds J. X. Jongejan and J. A. Duine, pp. 87±96. Kluwer Academic Publishers, Dordrecht.

Inoue, T., Sunagawa, M., Mori, A., Imai, C., Fukuda, M., Takagi, M., Yano, K., 1989. Cloning and sequencing of the gene encoding

the 72-Kilodalton dehydrogenase subunit of alcohol dehydrogenase from Acetobacter aceti. J. Bacteriol. 171, 3115±3122.

Inoue, T., Sunagawa, M., Mori, A., Imai, C., Fukuda, M., Takagi, M., Yano, K., 1990. Possible Functional Domains in a

Quinoprotein Alcohol Dehydrogenase from Acetobacter aceti. J. Ferment. Bioeng. 70, 58±60.

Ito, N., Phillips, S. E. V., Yadav, K. D. S., Knowles, P. F., 1994. Crystal structure of a free radical enzyme, galactose oxidase. J. Mol.

Biol. 238, 794±814.

Itoh, S., Kawakami, H., Fukuzumi, A., 1997. Modeling of the chemistry of quinoprotein methanol dehydrogenase, oxidation of

methanol by calcium complex of coenzyme PQQ via addition-elimination mechanism. J. Am. Chem. Soc. 119, 439±440.

Janes, S. M., Mu, D., Wemmer, D., Smith, A. J., Kaur, S., Maltby, D., Buringame, A. L., Klinman, J. P., 1990. A new redox cofactor

in eucaryotic enzymes: 6-hydroxydopa at the active site of bovine serum amine oxidase. Science 248, 981±987.

Kao, P. N., Karlin, A., 1986. Acetylcholine receptor binding site contains a disulphide cross-link between adjacent half-cystinyl resi-

dues. J. Biol. Chem. 261, 8085±8088.

Klinman, J. P. (1995) Quinoproteins; Methods in Enzymology. Academic Press, New York.

Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., Sigler, P. B., 1996. The 2.0 Angstrom crystal structure of a het-

erotrimeric G protein. Nature 379, 311±319.

Li, J., Brick, P., O'Hare, M. C., Skarzynski, T., Lloyd, L. F., Curry, V. A., Clark, I. M., Bigg, H. F., Hazleman, B. L., Cawston, T. E.,

Blow, D. M., 1995. Structure of a full-length porcine synovial collagenase reveals a C-terminal domain containing a clacium-linked,

four bladed propeller. Structure 3, 541±549.

Matsushita, K., Shinagawa, E., Inoue, T., Adachi, O., Ameyama, M., 1986. Immunological evidence for two types of PQQ-dependent

D-glucose dehydrogenase in bacterial membranes and the location of the enzyme in Escherichia coli. FEMS. Microbiol. Letts. 37,

141±144.


Matsushita, K., Shinagawa, E., Adachi, O., Ameyama, M., 1989. Reactivity with ubiquinone of quinoprotein D-glucose dehydrogen-

ase from Gluconobacter suboxydans. J. Biochem. 105, 633±637.

Matsushita, K., Toyama, H., Adachi, O., 1994. Respiratory chains and bioenergetics of acetic acid bacteria. Adv. Microbial. Physiol.

36, 247±301.

Matsushita, K. and Adachi, O. (1993) Bacterial quinoproteins glucose dehdyrogenase and alcohol dehydrogenase. In Principles and

application of quinoproteins, ed. V. L. Davidson, pp. 47±63. Marcel Dekker Inc, New York.

McIntire, W. S., Wemmer, D. E., Chistoserdov, A., Lidstrom, M. E., 1991. A new cofactor in a prokaryotic enzymeÐtryptophan tryp-

tophylquinone as the redox prosthetic group in methylamine dehydrogenase. Science 252, 817±824.

Mez, H., 1974. Cyclo-L-cystine.acetic acid. Cryst. Struct. Comm. 3, 657±660.

Murzin, A. G., 1992. Structural principles for the propellor assembly of B-sheets: the preference for seven-fold symmetry. Proteins 14,

191±201.

Nunn, D. N., Anthony, C., 1988. The nucleotide sequence and deduced amino acid sequence of the cytochrome cL gene of

Methylobacterium extorquens AM1: a novel class of c-type cytochromes. Biochem. J. 256, 673±676.

Ramachandran, G. N., Sasisekharan, V., 1968. Conformation of polypeptides and proteins. Adv. Protein Chem. 23, 283±437.

Richardson, I. W., Anthony, C., 1992. Characterization of mutant forms of the quinoprotein methanol dehydrogenase lacking an

essential calcium ion. Biochem. J. 287, 709±715.

Salisbury, S. A., Forrest, H. S., Cruse, W. B. T., Kennard, O., 1979. A novel coenzyme from bacterial primary alcohol dehydrogenases.

Nature 280, 843±844.

Schiering, N., Kabsch, W., Moore, M. J., Distefano, M. D., Walsh, C. T., Pai, E. F., 1991. Structure of the detoxi®cation catalyst

mercuric ion reductase from Bacillus sp. strain RC607. Nature 352, 168±172.

Schrover, J. M. J., Frank, J., Vanwielink, J. E., Duine, J. A., 1993. Quaternary structure of quinoprotein ethanol dehydrogenase from

Pseudomonas aeruginosa and its reoxidation with a novel cytochrome-c from this organism. Biochem. J. 290, 123±127.

Sondek, J., Bohm, A., Lambright, D. G., Hamm, H. E., Sigler, P. B., 1996. Crystal structure of a GA protein dimer at 2.1 Angstrom

resolution. Nature 379, 369±374.

Stoorvogel, J., Kraayveld, D. E., vanSluis, C. A., Jongejan, J. A., Devries, S., Duine, J. A., 1996. Characterization of the gene encoding

quinohaemoprotein ethanol dehydrogenase of Comamonas testosteroni. Eur. J. Biochem. 235, 690±698.

Sukumaran, D. K., Prorok, M., Lawrence, D. S., 1991. A molecular constraint that generates a cis peptide bond. J. Am. Chem. Soc.

113, 706±707.

Varghese, J. N., Laver, W. G., Colman, P. M., 1983. Structure of the in¯uenza virus glycogen antigen neuraminidase at 2.9 A resol-

ution. Nature 303, 35±40.

Vellieux, F. M. D., Huitema, F., Groendijk, H., Kalk, K. H., Frank, J., Jongejan, J. A., Duine, J. A., Petratos, K., Drenth, J., Hol, W.

G. J., 1989. Structure of quinoprotein methylamine dehydrogenase at 2.25 A resolution. EMBO. J. 8, 2171±2178.

Wang, S. X., Mure, M., Medzihradszky, K. F., Burlingame, A. L., Brown, D. E., Dooley, D. M., Smith, A. J., Kagan, H. M., Klinman,

J. P., 1996 LTQ. Science 273, 1078±1084.

White, S., Boyd, G., Mathews, F. S., Xia, Z. X., Dai, W. W., Zhang, Y. F., Davidson, V. L., 1993. The active site structure of the

calcium-containing quinoprotein methanol dehydrogenase. Biochemistry 32, 12955±12958.

Xia, Z., Dai, W., Zhang, Y., White, S. A., Boyd, G. D., Mathews, F. S., 1996. Determination of the gene sequence and the three-dimen-

sional structure at 2 angstrom resolution of methanol dehydrogenae from Methylophilus W3A1. J. Mol. Biol. 259, 480±501.

Xia, Z. X., Dai, W. W., Xiong, J. P., Hao, Z. P., Davidson, V. L., White, S., Mathews, F. S., 1992. The 3-dimensional structures of

methanol dehydrogenase from 2 methylotrophic bacteria at 2.6-angstrom resolution. J. Biol. Chem. 267, 22289±22297.


Documents

The structure and function of the PQQ-containing quinoprotein dehydrogenases